ONCOLOGY LETTERS 10: 1343-1349, 2015
Metformin inhibits growth of lung adenocarcinoma cells by inducing apoptosis via the mitochondria‑mediated pathway JUNLING WANG1, QIULING GAO2, DECUI WANG1, ZHIQIANG WANG1 and CHUN HU3 1
Department of Medical Oncology, Binzhou Tuberculosis Control Center, Binzhou Medical College; 2 Department of Radiology, Binzhou People's Hospital, Binzhou, Shandong 250000; 3 Department of Oncology, RuiJin Hospital, Shanghai JiaoTong University, Shanghai 200001, P.R. China Received May 20, 2014; Accepted January 29, 2015 DOI: 10.3892/ol.2015.3450 Abstract. Metformin is commonly used to treat type II diabetes, although it may also reduce the risk of cancer and improve the associated prognosis. However, its mode of action in cancer remains unclear. The present study evaluated the effects of metformin on lung adenocarcinoma A549 cells and identified molecular mechanisms of metformin activity. The A549 cells were treated with metformin at different concentrations and cell viability was assayed by using an MTT assay. The cell cycle and the apoptosis rate were assayed by flow cytometry. Nude mice were transplanted with A549 cells and the tumor growth inhibition rate was detected. Once the A549 cells had been treated with 20 mM metformin for 48 h, the cell cycle was arrested in the G0/Gl phase and the apoptosis rate was 20.57±3.16%. The expression of the B‑cell lymphoma (Bcl)‑2 and Bcl‑extra large proteins was downregulated following metformin treatment, while Bax protein expression was significantly increased. Tumor size in the high‑dose metformin and cisplatin plus metformin groups was significantly smaller, and the inhibition rates were 41.3 and 72.9%, respectively, compared with the control group. These results indicated that metformin displays anticancer activity against lung adenocarcinoma by causing G1 arrest of the cell cycle and subsequent cell apoptosis through the mitochondria‑dependent pathway in A549 cells. Furthermore, it was found that metformin dramatically inhibited lung adenocarcinoma tumor growth in vivo. These data suggest that metformin may become a potential cytotoxic drug in the prevention and treatment of lung adenocarcinoma.
Correspondence to: Mr. Chun Hu, Department of Oncology,
RuiJin Hospital, Shanghai JiaoTong University, 197 RuiJin Road, Shanghai 200001, P.R. China E‑mail: [email protected]
Key
words: metformin, lung mitochondrion‑mediated pathway
adenocarcinoma,
apoptosis,
Introduction Lung adenocarcinoma is one of the main causes of cancer‑related mortality globally, accounting for nearly 30% of cancer‑related mortalities worldwide (1). The incidence of lung adenocarcinoma is rising all over the world due to the adoption of lifestyle choices that have an association with cancer, including physical inactivity and smoking. Although advances have been made with regard to early diagnosis and treatment modalities, the prognosis for affected patients remains poor, with a five‑year survival rate of only ~15% (2). As a consequence, the prevention of lung cancer is a high priority and urgent efforts are required to identify measures, including drug treatment, which may effectively reduce the risk of lung cancer. Chemotherapy is one of the best approaches for unresectable tumors, but the efficacy of current lung tumor chemotherapy is only modest and the requirement for an optimal lung adenocarcinoma treatment remains. Metformin, a biguanide drug, has been demonstrated to exert anticancer effects (3). The drug reduces the level of glucose by decreasing liver glucose production, thereby increasing fatty acid oxidation and glucose utilization. Notably, previous epidemiological studies suggested that patients with diabetes who were treated with metformin had a lower cancer‑related mortality rate and a lower incidence rate of cancer of any type when compared with patients who underwent other treatments (4‑7). Additionally, metformin was shown to prevent the induction of carcinogen‑induced pancreatic cancer in hamsters that were maintained on high‑fat diets (8). The drug was also shown to inhibit the growth of breast and colon carcinoma cells (9,10). Collated evidence from a number of clinical studies has recently been published in a meta‑analysis (11). However, the precise mechanisms involved remain incompletely understood. The antitumor activity of metformin may be explained by two mechanisms. Firstly, metformin is able to decrease insulin resistance and lower the levels of circulating insulin by activating AMP‑activated protein kinase (AMPK), which causes decreased hepatic gluconeogenesis (10) and increased glucose uptake in the muscle. Secondly, metformin acts as an inhibitor of tumor growth, at least in part by upregulating the activity of AMPK and by downstream suppression of signaling through the mammalian target of rapamycin (mTOR) (12). Several other
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WANG et al: METFORMIN INHIBITS THE GROWTH OF LUNG ADENOCARCINOMA
potential mechanisms for the anticancer action of metformin have also been demonstrated, including the suppression of HER2 oncoprotein expression, the downregulation of cyclin D1 expression and p53 activation (9,13‑15). However, there have been few studies evaluating the potential utility of metformin in in vivo models of cancer, and the method by which metformin induces apoptosis remains unknown. The present study describes experiments that were performed to investigate the hypothesis that metformin exhibits direct anti‑proliferative actions on lung adenocarcinoma cells in vitro and in vivo. Materials and methods Chemicals and reagents. Metformin was obtained from Sigma‑Aldrich (St. Louis, MO, USA) and dissolved in phosphate‑buffered saline (PBS). Cell culture chemicals and materials were obtained from Invitrogen Life Technologies (Burlington, ON, Canada). Anti‑β‑actin, anti‑B‑cell lymphoma (Bcl)‑2, anti‑Bax and anti‑caspase‑3 were purchased from Cell Signaling Technology (Beverly, MA, USA). Horseradish peroxidase‑conjugated anti‑rabbit immnoglobulin (Ig)G, anti‑mouse IgG and enhanced chemiluminescence (ECL) reagents were obtained from Amersham Pharmacia Biotech (Piscataway, NJ, USA). Cell lines and culture conditions. The human lung adenocarcinoma A549 cells were obtained from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences (Shanghai, China). The A549 cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 2 mmol/l L‑glutamine, 100 U/ml penicillin and 100 µg/ml streptomycin at 37˚C, in an atmosphere of 5% CO2. The cells were passaged by 0.25% Trypsin‑EDTA when they reached 80% confluence. Cell proliferation assay. An MTT assay was used to evaluate the effect of metformin on the lung adenocarcinoma cells. Briefly, ~10,000 cells were seeded into 96‑well tissue culture plates and then treated with different doses (0, 5, 10, 20 and 50 mmol/l) of metformin for 24, 48 and 72 h, respectively. MTT reagent was then added to each well, and the cells were further incubated for 6 h. Absorbance was measured in an automated microplate reader (ELX 800; BioTek Instruments, Inc., Winooski, VT, USA) at 450 nm. Cell morphological analysis. The A549 cells were treated with 10.0 mM metformin or 0.1% dimethyl sulfoxide (control) for 48 h. The cells were then incubated with 10 µg/ml Hoechst 33342 and observed by fluorescence microscope (DMIRB; Leica, Wetzler, Germany). Flow cytometry. The A549 cells were starved of serum for 24 h and then treated with different doses (0, 10 and 20 mmol/l) of metformin for 48 h. The cells were then washed with PBS (pH 7.4) and fixed with 70% ice‑cold ethanol at 4˚C overnight. After fixation, the cells were stained with propidium iodide (PI) at 1 mg/ml for 30 min at room temperature. The cell cycle was analyzed by flow cytometry (FACScan; BD Biosciences, Franklin Lakes, NJ, USA).
For cell apoptosis detection, the apoptotic rate of A549 cells was analyzed using an Annexin Ⅴ‑fluorescein isothiocyanate (FITC) apoptosis detection kit (Nanjing KeyGen Biotech Co., Ltd., Nanjing, China). A total of 1x105 cells/well were seeded into six-well plates and cultured in DMEM at 37˚C overnight. Subsequent to starvation for 12 h, the cells were treated with different doses of metformin (0, 10 and 20 mmol/l) in complete medium for 48 h, digested with 2.5 mg/ml trypsin, washed twice with PBS and suspended with 300 µl binding buffer (Nanjing KeyGen Biotech Co., Ltd.). The cells were then incubated with 2 µl Annexin V and 5 µl PI for 15 min at room temperature, and the distribution of viable, early apoptotic, late apoptotic and necrotic cells was detected using a FACSCaliber flow cytometer (BD Biosciences). Cells that were negative for the Annexin Ⅴ‑FITC and PI were considered to be viable cells, cells that were positive for Annexin Ⅴ‑FITC, but negative for PI were considered to be early apoptotic cells, cells that were positive for Annexin Ⅴ‑FITC and PI were considered to be late apoptotic cells, while cells that were negative for both Annexin Ⅴ‑FITC and PI were considered to be necrotic. The sum of the early and late apoptotic cells constituted the total number of apoptotic cells, which was presented as the percentage of the total cells. Mitochondrial and cytosolic fractionation. The Cell Mitochondria Isolation kit (Beyotime Institute of Biotechnology, Haimen, China) was used to perform the isolation of the mitochondria and cytosol, according to the manufacturer's instructions. Samples of cytosol and mitochondria were dissolved in lysis buffer, and proteins were subjected to western blotting, respectively. Western blot analysis. The A549 cells were lysed in a radioimmunoprecipitation assay buffer (9.1 mM dibasic sodium phosphate, 1.7 mM monobasic sodium phosphate, 150 mM NaCl, 1% NP40, 0.5% sodium deoxycholate, 0.1% SDS, 0.2 mM sodium vanadate, 0.2 mM phenylmethylsulfonyl fluoride and 0.2 U/ml aprotinin). Clarified protein lysates (50 g) were resolved electrophoretically on denaturing SDS‑polyacrylamide gels (10%), and transferred to nitrocellulose membranes. The membranes were then blocked with 1% bovine serum albumin at room temperature for 1 h and then incubated with the indicated specific primary antibodies for 3 h. Proteins were visualized with Horseradish peroxidase (HRP)‑conjugated secondary antibodies. To corroborate equal loading, membranes were stripped and reprobed using an antibody specific for β‑actin. Finally, antigen‑antibody complexes were detected using the ECL system. A549 tumor xenograft. A total of 6x10 6 A549 cells were injected into the right flank of 30 BALB/c nude mice (supplied by the Experimental Animal Department of Binzhou medical College, Shandong, China). Seven days later, 25 mice with tumors ~100 mm 3 in size were randomly distributed into the following five groups: Control group (PBS), low‑dose metformin (40 mg/kg/day) group, high‑dose metformin (200 mg/kg/day) group, cisplatin (5 mg/kg/day) group and metformin (40 mg/kg/day) plus cisplatin (5 mg/kg/day) group. Tumor volume (mean values and 95% confidence intervals) was measured every three days after the initial injection. After
ONCOLOGY LETTERS 10: 1343-1349, 2015
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Figure 1. Effects of metformin on the proliferation of A549 cells. (A) Metformin inhibited the proliferation of the cells in a dose‑ and time‑dependent manner. The A549 cells were exposed to increasing doses of metformin for 24, 48 and 72 h respectively, and cell proliferation was measured using the MTT assay. (B) The addition of metformin potentiated the cytotoxicity induced by cisplatin. A549 cells were cultured for 48 h in the presence of increasing amounts of cisplatin with and without metformin, and cell proliferation was measured using the MTT assay. The results are shown as the mean ± standard deviation of triplicate experiments. Ctl, control; cis, cisplatin.
Figure 2. Metformin blocks the cell cycle in the G 0/G1 phase. Flow cytometry analysis of proliferating A549 cells 48 h after the treatment with metformin (10 and 20 mM). Fractions of cells in the G 0/G1, S and G2/M phases of the cell cycle are indicted. Untreated cells were used as controls. *P
Metformin inhibits growth of lung adenocarcinoma cells by inducing apoptosis via the mitochondria‑mediated pathway JUNLING WANG1, QIULING GAO2, DECUI WANG1, ZHIQIANG WANG1 and CHUN HU3 1
Department of Medical Oncology, Binzhou Tuberculosis Control Center, Binzhou Medical College; 2 Department of Radiology, Binzhou People's Hospital, Binzhou, Shandong 250000; 3 Department of Oncology, RuiJin Hospital, Shanghai JiaoTong University, Shanghai 200001, P.R. China Received May 20, 2014; Accepted January 29, 2015 DOI: 10.3892/ol.2015.3450 Abstract. Metformin is commonly used to treat type II diabetes, although it may also reduce the risk of cancer and improve the associated prognosis. However, its mode of action in cancer remains unclear. The present study evaluated the effects of metformin on lung adenocarcinoma A549 cells and identified molecular mechanisms of metformin activity. The A549 cells were treated with metformin at different concentrations and cell viability was assayed by using an MTT assay. The cell cycle and the apoptosis rate were assayed by flow cytometry. Nude mice were transplanted with A549 cells and the tumor growth inhibition rate was detected. Once the A549 cells had been treated with 20 mM metformin for 48 h, the cell cycle was arrested in the G0/Gl phase and the apoptosis rate was 20.57±3.16%. The expression of the B‑cell lymphoma (Bcl)‑2 and Bcl‑extra large proteins was downregulated following metformin treatment, while Bax protein expression was significantly increased. Tumor size in the high‑dose metformin and cisplatin plus metformin groups was significantly smaller, and the inhibition rates were 41.3 and 72.9%, respectively, compared with the control group. These results indicated that metformin displays anticancer activity against lung adenocarcinoma by causing G1 arrest of the cell cycle and subsequent cell apoptosis through the mitochondria‑dependent pathway in A549 cells. Furthermore, it was found that metformin dramatically inhibited lung adenocarcinoma tumor growth in vivo. These data suggest that metformin may become a potential cytotoxic drug in the prevention and treatment of lung adenocarcinoma.
Correspondence to: Mr. Chun Hu, Department of Oncology,
RuiJin Hospital, Shanghai JiaoTong University, 197 RuiJin Road, Shanghai 200001, P.R. China E‑mail: [email protected]
Key
words: metformin, lung mitochondrion‑mediated pathway
adenocarcinoma,
apoptosis,
Introduction Lung adenocarcinoma is one of the main causes of cancer‑related mortality globally, accounting for nearly 30% of cancer‑related mortalities worldwide (1). The incidence of lung adenocarcinoma is rising all over the world due to the adoption of lifestyle choices that have an association with cancer, including physical inactivity and smoking. Although advances have been made with regard to early diagnosis and treatment modalities, the prognosis for affected patients remains poor, with a five‑year survival rate of only ~15% (2). As a consequence, the prevention of lung cancer is a high priority and urgent efforts are required to identify measures, including drug treatment, which may effectively reduce the risk of lung cancer. Chemotherapy is one of the best approaches for unresectable tumors, but the efficacy of current lung tumor chemotherapy is only modest and the requirement for an optimal lung adenocarcinoma treatment remains. Metformin, a biguanide drug, has been demonstrated to exert anticancer effects (3). The drug reduces the level of glucose by decreasing liver glucose production, thereby increasing fatty acid oxidation and glucose utilization. Notably, previous epidemiological studies suggested that patients with diabetes who were treated with metformin had a lower cancer‑related mortality rate and a lower incidence rate of cancer of any type when compared with patients who underwent other treatments (4‑7). Additionally, metformin was shown to prevent the induction of carcinogen‑induced pancreatic cancer in hamsters that were maintained on high‑fat diets (8). The drug was also shown to inhibit the growth of breast and colon carcinoma cells (9,10). Collated evidence from a number of clinical studies has recently been published in a meta‑analysis (11). However, the precise mechanisms involved remain incompletely understood. The antitumor activity of metformin may be explained by two mechanisms. Firstly, metformin is able to decrease insulin resistance and lower the levels of circulating insulin by activating AMP‑activated protein kinase (AMPK), which causes decreased hepatic gluconeogenesis (10) and increased glucose uptake in the muscle. Secondly, metformin acts as an inhibitor of tumor growth, at least in part by upregulating the activity of AMPK and by downstream suppression of signaling through the mammalian target of rapamycin (mTOR) (12). Several other
1344
WANG et al: METFORMIN INHIBITS THE GROWTH OF LUNG ADENOCARCINOMA
potential mechanisms for the anticancer action of metformin have also been demonstrated, including the suppression of HER2 oncoprotein expression, the downregulation of cyclin D1 expression and p53 activation (9,13‑15). However, there have been few studies evaluating the potential utility of metformin in in vivo models of cancer, and the method by which metformin induces apoptosis remains unknown. The present study describes experiments that were performed to investigate the hypothesis that metformin exhibits direct anti‑proliferative actions on lung adenocarcinoma cells in vitro and in vivo. Materials and methods Chemicals and reagents. Metformin was obtained from Sigma‑Aldrich (St. Louis, MO, USA) and dissolved in phosphate‑buffered saline (PBS). Cell culture chemicals and materials were obtained from Invitrogen Life Technologies (Burlington, ON, Canada). Anti‑β‑actin, anti‑B‑cell lymphoma (Bcl)‑2, anti‑Bax and anti‑caspase‑3 were purchased from Cell Signaling Technology (Beverly, MA, USA). Horseradish peroxidase‑conjugated anti‑rabbit immnoglobulin (Ig)G, anti‑mouse IgG and enhanced chemiluminescence (ECL) reagents were obtained from Amersham Pharmacia Biotech (Piscataway, NJ, USA). Cell lines and culture conditions. The human lung adenocarcinoma A549 cells were obtained from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences (Shanghai, China). The A549 cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 2 mmol/l L‑glutamine, 100 U/ml penicillin and 100 µg/ml streptomycin at 37˚C, in an atmosphere of 5% CO2. The cells were passaged by 0.25% Trypsin‑EDTA when they reached 80% confluence. Cell proliferation assay. An MTT assay was used to evaluate the effect of metformin on the lung adenocarcinoma cells. Briefly, ~10,000 cells were seeded into 96‑well tissue culture plates and then treated with different doses (0, 5, 10, 20 and 50 mmol/l) of metformin for 24, 48 and 72 h, respectively. MTT reagent was then added to each well, and the cells were further incubated for 6 h. Absorbance was measured in an automated microplate reader (ELX 800; BioTek Instruments, Inc., Winooski, VT, USA) at 450 nm. Cell morphological analysis. The A549 cells were treated with 10.0 mM metformin or 0.1% dimethyl sulfoxide (control) for 48 h. The cells were then incubated with 10 µg/ml Hoechst 33342 and observed by fluorescence microscope (DMIRB; Leica, Wetzler, Germany). Flow cytometry. The A549 cells were starved of serum for 24 h and then treated with different doses (0, 10 and 20 mmol/l) of metformin for 48 h. The cells were then washed with PBS (pH 7.4) and fixed with 70% ice‑cold ethanol at 4˚C overnight. After fixation, the cells were stained with propidium iodide (PI) at 1 mg/ml for 30 min at room temperature. The cell cycle was analyzed by flow cytometry (FACScan; BD Biosciences, Franklin Lakes, NJ, USA).
For cell apoptosis detection, the apoptotic rate of A549 cells was analyzed using an Annexin Ⅴ‑fluorescein isothiocyanate (FITC) apoptosis detection kit (Nanjing KeyGen Biotech Co., Ltd., Nanjing, China). A total of 1x105 cells/well were seeded into six-well plates and cultured in DMEM at 37˚C overnight. Subsequent to starvation for 12 h, the cells were treated with different doses of metformin (0, 10 and 20 mmol/l) in complete medium for 48 h, digested with 2.5 mg/ml trypsin, washed twice with PBS and suspended with 300 µl binding buffer (Nanjing KeyGen Biotech Co., Ltd.). The cells were then incubated with 2 µl Annexin V and 5 µl PI for 15 min at room temperature, and the distribution of viable, early apoptotic, late apoptotic and necrotic cells was detected using a FACSCaliber flow cytometer (BD Biosciences). Cells that were negative for the Annexin Ⅴ‑FITC and PI were considered to be viable cells, cells that were positive for Annexin Ⅴ‑FITC, but negative for PI were considered to be early apoptotic cells, cells that were positive for Annexin Ⅴ‑FITC and PI were considered to be late apoptotic cells, while cells that were negative for both Annexin Ⅴ‑FITC and PI were considered to be necrotic. The sum of the early and late apoptotic cells constituted the total number of apoptotic cells, which was presented as the percentage of the total cells. Mitochondrial and cytosolic fractionation. The Cell Mitochondria Isolation kit (Beyotime Institute of Biotechnology, Haimen, China) was used to perform the isolation of the mitochondria and cytosol, according to the manufacturer's instructions. Samples of cytosol and mitochondria were dissolved in lysis buffer, and proteins were subjected to western blotting, respectively. Western blot analysis. The A549 cells were lysed in a radioimmunoprecipitation assay buffer (9.1 mM dibasic sodium phosphate, 1.7 mM monobasic sodium phosphate, 150 mM NaCl, 1% NP40, 0.5% sodium deoxycholate, 0.1% SDS, 0.2 mM sodium vanadate, 0.2 mM phenylmethylsulfonyl fluoride and 0.2 U/ml aprotinin). Clarified protein lysates (50 g) were resolved electrophoretically on denaturing SDS‑polyacrylamide gels (10%), and transferred to nitrocellulose membranes. The membranes were then blocked with 1% bovine serum albumin at room temperature for 1 h and then incubated with the indicated specific primary antibodies for 3 h. Proteins were visualized with Horseradish peroxidase (HRP)‑conjugated secondary antibodies. To corroborate equal loading, membranes were stripped and reprobed using an antibody specific for β‑actin. Finally, antigen‑antibody complexes were detected using the ECL system. A549 tumor xenograft. A total of 6x10 6 A549 cells were injected into the right flank of 30 BALB/c nude mice (supplied by the Experimental Animal Department of Binzhou medical College, Shandong, China). Seven days later, 25 mice with tumors ~100 mm 3 in size were randomly distributed into the following five groups: Control group (PBS), low‑dose metformin (40 mg/kg/day) group, high‑dose metformin (200 mg/kg/day) group, cisplatin (5 mg/kg/day) group and metformin (40 mg/kg/day) plus cisplatin (5 mg/kg/day) group. Tumor volume (mean values and 95% confidence intervals) was measured every three days after the initial injection. After
ONCOLOGY LETTERS 10: 1343-1349, 2015
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Figure 1. Effects of metformin on the proliferation of A549 cells. (A) Metformin inhibited the proliferation of the cells in a dose‑ and time‑dependent manner. The A549 cells were exposed to increasing doses of metformin for 24, 48 and 72 h respectively, and cell proliferation was measured using the MTT assay. (B) The addition of metformin potentiated the cytotoxicity induced by cisplatin. A549 cells were cultured for 48 h in the presence of increasing amounts of cisplatin with and without metformin, and cell proliferation was measured using the MTT assay. The results are shown as the mean ± standard deviation of triplicate experiments. Ctl, control; cis, cisplatin.
Figure 2. Metformin blocks the cell cycle in the G 0/G1 phase. Flow cytometry analysis of proliferating A549 cells 48 h after the treatment with metformin (10 and 20 mM). Fractions of cells in the G 0/G1, S and G2/M phases of the cell cycle are indicted. Untreated cells were used as controls. *P
